NADPH oxidase, which produces superoxide, is an important bactericidal product of neutrophils and critically important in host defense. The oxidase complex includes cytochrome b559 which is a heterodimer of p22phox and gp91phox as well as cytosolic proteins (p47phox, p67phox and Rax1/2 which assemble into the oxidase upon activation. Although expression of the oxidase has been thought to be myeloid specific, new evidence indicates that similar proteins and oxidase activity occur in other cell types. In this regard, the applicants have recently cloned the gene NOH-1 which encodes a gp91phox homologue expressed in non-myeloid cells, especially colonic epithelium. The predicted protein has conserved sites for binding of heme, FAD and NADPH and appears to be able to generate superoxide. This suggests the existence of a family of mammalian plasma membrane flavo-heme oxidoreductases which may share, with phagocyte NADPH oxidase, similar biochemical and molecular mechanisms but exhibit tissue-specific function, regulation and roles in the host defense system. To explore this prospect, the specific aims of the present grant are to: (1) characterize the biochemical features and molecular mechanisms of the oxidoreductase activity of gp91phox and the homologous NOH-1, as well as define cofactors, substrates, structural requirements and mechanisms of enzyme activation, (2) determine the possible role of NOH-1 as an antimicrobial host defense system in colon epithelium and (3) examine the regulation of expression of NADPH oxidase genes, with an emphasis on the molecular bases for specificity of expression in myeloid vs. non-myeloid tissues and the response to inflammatory stimuli and cell maturation.